牙龈卟啉单胞菌W83牙龈蛋白酶的提取、鉴定及对人成骨细胞增殖、凋亡的影响

The extraction, identification of gingipains from Porphyromonas gingivalis W83 and its effects on proliferation and apoptosis of human osteoblasts

目的研究提纯并鉴定牙龈卟啉单胞菌(P.g)W83牙龈蛋白酶的方法,探讨牙龈蛋白酶对人成骨细胞系hFOB增殖及凋亡的影响。方法丙酮沉淀P.gW83上清,经重悬、透析、超滤提取牙龈蛋白酶;SDS-聚丙烯酰胺凝胶电泳法(SDS-PAGE)分离牙龈蛋白酶条带;基质辅助激光解吸电离飞行时间质谱/源后衰变法(MALDI-TOF-MS/MS+MS)鉴定牙龈蛋白酶提取物;底物发色法测定牙龈蛋白酶活性;牙龈蛋白酶与人成骨细胞系hFOB1.19共培养,4′,6-二脒基-2-苯基吲哚(DAPI)或CCK-8检测细胞凋亡或增殖。结果 P.gW83牙龈蛋白酶提取物经SDS-PAGE分离出50kDa蛋白条带,MALDI-TOF-MS/MS+MS鉴定其为精氨酸特异性半胱氨酸蛋白酶(Rgps)。底物发色法测定Rgps、赖氨酸特异性半胱氨酸蛋白酶(Kgp)活性分别为75.62、10.51U/L,5mmol/L半胱氨酸蛋白酶抑制剂TLCK对Rgps、Kgp活性抑制分别为99.51%、99.00%(P<0.01)。牙龈蛋白酶(Rgps活性15.12U/L、Kgp活性2.10U/L)与hFOB共培养8h可诱导细胞发生凋亡,出现典型的细胞核染色质凝聚,且随时间增加hFOB凋亡率逐渐上升,16～24h达到高峰。CCK-8法检测显示,牙龈蛋白酶呈时间依赖性抑制hFOB增殖。结论丙酮沉淀P.gW83上清,经重悬、透析、超滤提纯的牙龈蛋白酶主要成分是Rgps;底物发色法测定Rgps/Kgp活性,方法稳定、可靠;牙龈蛋白酶抑制hFOB增殖且促进凋亡。

Objective To purify and identify gingipains from Porphyromonas gingivalis W83 （P.g W83）, and to investigate the effects of gingipains on proliferation and apoptosis of human osteoblast hFOB 1.19. Methods Gingipains were extracted from supernatant of P.g W83 using acetone precipitation, followed by resuspension, dialysis and uhrafihration. Purified gingipains separated by SDS-PAGE were characterized by peptide mass fingerprinting and peptide fragmentation fingerprinting using MALDI-TOF-MS/MS ＋MS. Activity of gingipains was determined by chromogenic substrate BAPNA/ALNA. Human osteoblasts were treated with purified gingipains for 8, 16, 54, 36 and 48 h. Proliferation activity was determined by CCK-8 assay and apoptosis was examed by DAPI staining. Results Gingipains extracted from P.g W83 were separated by SDS-PAGE, resuhing in a 50 kDa sharp band. The 50 kDa protein was characterized as arginine-specific gingipain using MALDI-TOF-MS/ MS ＋MS. Enzymatic activities of purified gingipains were 75.62 and 10.51 U/L for Rgps or Kgp respectively. Gingipains activities were fully blocked by 5 mmol/L TLCK, a cysteine protease inhibitor. DAPI staining revealed that purified gingipains （15.12 U/L Rgps activity and 2.10 U/L Kgp activity） induced osteoblast apoptosis from 8 h and peaked from 16 to 24 h. CCK-8 assay showed that gingipainstime-dependently inhibited the proliferation of osteoblasts. Conclusions Gingipains extracted from P.g W83 were characterized as arginine-specific gingipain. Activity of gingipains was determined by chromogenic substrate method, which is stable and reliable. Gingipains inhibited proliferation and induced apoptosis of human osteoblasts.